Crystal structure of the Sema-PSI extracellular domain of human RON receptor tyrosine kinase.

Human RON (Recepteur d'Origine Nantais) receptor tyrosine kinase is a cell surface receptor for Macrophage Stimulating Protein (MSP). RON mediates signal transduction pathways that regulate cell adhesion, invasion, motility and apoptosis processes. Elevated levels of RON and its alternatively spliced variants are implicated in the progression and metastasis of tumor cells. The binding of MSP α/β heterodimer to the extracellular region of RON receptor induces receptor dimerization and activation by autophosphorylation of the intracellular kinase domains. The ectodomain of RON, containing the ligand recognition and dimerization domains, is composed of a semaphorin (Sema), Plexins-Semaphorins-Integrins domain (PSI), and four Immunoglobulins-Plexins-Transcription factor (IPT) domains. High affinity association between MSP and RON is mediated by the interaction between MSP β-chain and RON Sema, although RON activation requires intact RON and MSP proteins. Here, we report the structure of RON Sema-PSI domains at 1.85 Å resolution. RON Sema domain adopts a seven-bladed β-propeller fold, followed by disulfide bond rich, cysteine-knot PSI motif. Comparison with the homologous Met receptor tyrosine kinase reveals that RON Sema-PSI contains distinguishing secondary structural features. These define the receptors' exclusive selectivity towards their respective ligands, RON for MSP and Met for HGF. The RON Sema-PSI crystal packing generates a homodimer with interface formed by the Sema domain. Mapping of the dimer interface using the RON homology to Met, MSP homology to Hepatocyte Growth Factor (HGF), and the structure of the Met/HGF complex shows the dimer interface overlapping with the putative MSPβ binding site. The crystallographically determined RON Sema-PSI homodimer may represent the dimer assembly that occurs during ligand-independent receptor activation and/or the inhibition of the constitutive activity of RONΔ160 splice variant by the soluble RON splice variant, RONΔ85

Data collection and refinement statistics.

a<p>The values in parentheses are for the highest resolution shell, 1.85 Å.</p>b<p><i>R_merge</i>  = ∑<i>_hkl</i> [(∑<i>_j</i> | <i>I_j</i> – < <i>I</i> > | )/∑<i>_j</i> | <i>I_j</i> | ].</p>c<p><i>R_factor</i>  = ∑<i>_hkl</i> | |<i>F_o</i>| – |<i>F_c</i>| |/∑<i>_hkl</i> |<i>F_o</i>|, where <i>F_o</i> and <i>F_c</i> are the observed and calculated structure factors, respectively.</p>d<p><i>R_free</i> is computed from randomly selected 2,628 reflections and omitted from the refinement.</p

Crystal packing generates a RON homodimer interface that overlaps with the putative MSPβ binding site predicted based on the Met/HGF structure.

<p>(A) Left panel: Surface and ribbon representations of symmetry-related RON Sema-PSI molecules. Right panel: Close-up view of the interface and the molecules rotated by ∼90°. (B) Surface and ribbon representation of the modeled RON Sema-PSI/MSPβ complex derived based on the free MSPβ (PDB code 2ASU) and RON Sema-PSI structures superposed onto the structure of Met Sema-PSI/HGFβ (PDB entry 1SHY). The molecular surfaces of RON Sema-PSI (blue) and MSPβ (pink) are shown in transparent colors and secondary structural elements are shown in ribbon representation. (C) Stereoscopic representations of the RON Sema homodimer interface residues generated by crystal packing. The two subunits are colored gray and sky blue. Selected amino acids are colored in the atomic color scheme: red, oxygen; blue, nitrogen; dark yellow, sulfur; bright yellow, acetate carbon.</p

Structure-based sequence alignments of human RON and Met Sema-PSI.

<p>Residues are colored as follows: Identical residues (red), and conservatively replaced residues (blue) are boxed. Cysteines are colored gold. Matching colored symbols indicate pairs of cysteines that form disulfide bonds. Secondary structure units of RON and Met are labeled. The blue dots above the RON Sema residues indicate amino acids at the symmetry-related RON Sema-Sema interface as discussed in the text. The blue dots below the Met Sema sequence show residues that contact the HGFβ ligand (PDB code 1SHY) (Stamos et al., 2004). This figure was prepared with ESPript (espript.ibcp.fr/Espript/).</p

Expression of

Labeling of proteins with deuterium is an essential tool in overcoming size limitations in the application of nuclear magnetic resonance (NMR) spectroscopy to proteins larger than 30 kilodaltons (kDa). A non-originator antigen-binding fragment (Fab) of NIST RM 8671 NISTmAb, so called yNIST-Fab, is a ~ 50 kDa protein, with 5 native disulfide linkages, that can be expressed in properly folded form in methylotrophic Komagataella phaffii (formerly Pichia pastoris). Further, the K. phaffii host can support the production of perdeuterated yNIST-Fab which is necessary to obtain well-resolved TROSY-based tripleresonance NMR spectra for chemical shift assignment of the peptide backbone resonances. Here, we examined growth conditions and effects of media composition to maximize biomass generation and expression yield of the 2H, 13C, 15N-enriched NIST-Fab fragment. Triple-labeled yNIST-Fab with ~93% deuteration reduced the 1HN, 15N and 13C-linewidths in the NMR spectra, allowing sequential NMR assignment of backbone resonance a key step toward sequence-specific structural and dynamic studies of Fab fragments and intact antibodies

Structure of the Sema-PSI domains of human RON receptor tyrosine kinase.

<p>(A) Ribbon representation of RON Sema-PSI, viewed down the β-propeller, with the color ramped from blue at the N-terminus to red at the C-terminus. The blades are numbered and the antiparallel β-strands of each blade are labeled A–D from the inner to the outermost strands. Disulfide bonds are shown in red and the N-linked oligosaccharide is shown as stick models with the atomic color scheme: Green – carbon, red – oxygen, blue – nitrogen. (B) Surface rendering of the top and bottom surfaces of RON Sema-PSI, represented by electrostatistic potential from red (−10 k_bT/e_c) to blue (10 k_bT/e_c), as generated by PyMol. The left panel represents the top surface using the same orientation as in (A). The right panel corresponds to the opposite or bottom β-propeller surface.</p